The goal of chemotherapy of mycobacterial infections is to stop the worsening of the disease, to convert secretions to a noninfectious state by killing the bacilli if possible, and to allow healing of gross pathological damage. Tuberculosis is not cured by present drugs in the strict sense of the word, even though relapse rates can be minimized by optimal treatment. Characteristically, tubercle bacilli are slowly disposed of or killed by the body after the best available chemotherapy. The widespread use of isoniazid for example has been accompanied by the development of resistant strains with the result that current drugs may fail to eradicate the mycobacterial infections. It is therefore important to develop new drugs with different mechanisms of antimycobacterial action. However, these agents need not be those associated with highest potency on a dosage basis for general utility.
Optimal antituberculous therapy requires the use of several drugs in combination from the outset of therapy. Mycobacterial populations contain some spontaneous mutants which are resistant to drugs even prior to exposure. The frequency of such mutations can vary between 1 in less than 100 to 1 in greater than 10,000, depending upon the drug. Single drug therapy can inhibit the majority of organisms in an infected site, yet permit, and in fact encourage, uncontrolled growth of the resistant mutants. Early combination therapy with at least two drugs is the preferable method of preventing emergence of large resistant populations in the original tuberculous cavities. (Antimycobacterial agents are discussed at length in Medicinal Chemistry, Part I, Alfred Burger, ed. (Wiley-Interscience, N.Y. 1970), Chapter 19.)
Some therapeutic agents are most valuable for their ability to suppress emergence of resistance during combination therapy. An example is p-aminosalicylic acid, which can delay development of streptomycin resistance. See Burger, p. 429. Thus, anti-mycobacterial agents can be important not only for their own efficacy against susceptible organisms but for their ability to enhance effectiveness of other agents by controlling emergence of resistant populations, for example populations resistant to pyrazinamide, which is a major drug used in the therapy of tuberculosis. The synthesis of pyrazinamide was described by Kushner et al, J. Am. Chem. Soc. 74:3617 (1952), and the compound was patented in 1954 as a tuberculostatic agent. Williams, U.S. Pat. No. 2,677,641. When pyrazinamide is used alone resistance develops quickly, and for this reason it is usually administered in combination with other drugs such as isoniazid. Another disadvantage of pyrazinamide is its hepatotoxicity.
Although the precise mechanism of action of pyrazinamide is not known, it is hypothesized that the compound is acted upon by an amidase in the Mycobacterial cells, releasing pyrazinoic acid as the active component of the compound. Pyrazinamide is only active against Mycobacterium (M.) tuberculosis. It is not active against the closely related organism M. bovis or other mycobacteria.
It has been suggested that resistance to pyrazinamide is based on a decreased level of the nicotinamidase in resistant organisms. We hypothesized that if the level of the amidase was important in resistance to this compound, one might develop a series of pyrazinoic acid esters which would circumvent this mechanism of resistance because they would require an esterase rather than an amidase for their activation. Evaluation of several commercially available nicotinic acid esters suggested that pyrazinoic acid esters might be effective against pyrazinamide-resistant M. tuberculosis and M. bovis.
There is little or no support in the prior art for using pyrazinoic acid esters as tuberculostatic agents. U.S. Pat. No. 2,646,431 issued to Dalalian and Kushner covered pyrazine derivatives and methods of preparation. One such group of derivatives, thiolpyrazinoates, showed bacteriostatic and bacteriocidal properties against human tubercle bacillus. However, the specification states that in general, pyrazine monocarboxylic acid and derivatives such as esters do not possess bacteriostatic or bacteriocidal properties.
In 1954 Kushner et al, J. Am. Chem. Soc. 77:1152-1155, reported the use of ethyl mercaptan and related compounds in experimental treatment of tuberculosis. Isopropyl thiopyrazinoate applied subcutaneously exhibited activity in a standardized mouse test. However, the authors attributed this activity to the release of ethyl mercaptan, not to the pyrazinoyl residue. Brown et al, J. Am. Chem. Soc. 76:3860 (1954) also reported that ethyl mercapto compounds had antituberculosis activity, thus supporting the Kushner et al. assertion that the activity of ethyl thiolpyrazinoate was due to ethyl mercaptan and not the pyrazinoyl residue. The only suggestion that pyrazinoic acid esters might have some value in tuberculosis therapy is found in Solomons and Spoerri, J. Am. Chem. Soc. 75:679 (1953). In the course of evaluating esters of pyrazinoic and pyrazine-2,3-dicarboxylic acids as local anaesthetics, the authors learned of the effectiveness of pyrazinamide as a tuberculostatic agent. The authors tested their anaesthetic compounds for in vitro activity against Mycobacterium tuberculosis H37RV and reported that a few were active, including N,N-dimethyl-2-aminoethyl pyrazinoate. No further work appears to have been done with this compound, however. In addition, effectiveness against other mycobacteria, including pyrazinamide-resistant M. tuberculosis, would not have been obvious on the basis of this isolated in vitro test.
In 1958, Suzuki et al, Takamine Kenkyusho Nempo 10:19-23, reported that the pyrazinoate ester of chloramphenicol was inactive against a number of bacteria including M. tuberculosis.